Several converging lines of evidence implicate Notch signaling as important in colorectal carcinogenesis and that increased Notch signaling occurs in the context of upregulated Wnt/?-catenin/TCF signaling, although its roles and impact on tumor phenotype are incompletely understood. We have developed evidence for increased Notch expression, and downstream target genes including hes and hey as well as four jointed box-1 (FJX1) and others in human colorectal cancers as compared with adenomas and with normal colon mucosa from gene expression arrays of human colorectal cancers. We have also developed evidence that shifting colon cancer cells from the mesenchymal phenotype to the more epithelial phenotype through restoration of the tumor suppressor, Smad4, in colon cancer cells is accompanied by downregulation of FJX1 a downstream target of Notch, thus suggesting that Smad4 expression may disrupt Notch signaling. Taken together, our preliminary findings suggest that interactions between Notch and TGF-?/BMP/Smad signaling have marked effects on determining colon cancer cell phenotype in a potentially regulated manner. In addition, we have found that inhibition of COX-2 activity with celecoxib in a clinical trial resulted in decreased mRNA expression of the Notch target, FJX1. Since COX-2 activity and its products such as PGE2 and other prostaglandins play important roles in cancer cell survival, invasion and angiogenesis, this may link Notch signaling to these important biological processes and to tumor progression. Central Hypothesis: Dysregulation of Notch signaling and overexpression of the Notch target gene, fjx1, contribute to colorectal carcinoma progression. Based on preliminary results, we further we hypothesize that Notch signaling in colorectal cancer is modulated by TGF- ?/BMP/Smad4 and Wnt signaling and that COX-2 enzyme activity enhances fjx1 expression and Notch activity. The following specific aims are designed to test the hypotheses: 1. To determine whether increased FJX1 expression is directly related to increased Notch signaling and determine the role of Notch signaling and FJX1 expression on colon cancer cell proliferation, apoptosis, invasion and metastasis. 2. To determine the effect of Wnt, TGF-?/BMP signaling and COX-2 activity on the level of Notch activity and FJX1 expression in colorectal cancer. 3. To determine the impact of upregulated Notch signaling and FJX1 expression on colorectal tumors in vivo. A long-term goal of this project is to exploit a better understanding of the roles of Notch signaling and FJX1 expression and their interactions with other pathways in colorectal cancer to identify novel therapeutic interventions for this lethal disease. [unreadable] [unreadable] [unreadable] [unreadable]